Pure Appl. Chem., Vol. 76, No. 10, pp. 1869–1886, 2004.
© 2004 IUPAC
Acid-base properties of purine residues and the
effect of metal ions:
Quantification of rare nucleobase tautomers*
Helmut Sigel
Department of Chemistry, Inorganic Chemistry, University of Basel,
Spitalstrasse 51, CH-4056 Basel, Switzerland
Abstract: The macro acidity constants valid for aqueous solutions of several adenine, guanine, and hypoxanthine derivatives are summarized. It is shown how the application of the
corresponding constants, e.g., for 7,9-dimethyladenine, allows a quantification of the intrinsic acidic properties of the (N1)H0/+ and (N7)H+ sites via micro acidity constants, and how
to use this information for the calculation of the tautomeric ratios regarding the monoprotonated species, that is, N7-N1H versus HN7-N1, meaning that in one isomer H+ is at the N1
site and in the other at N7. It is further shown that different metal ions coordinated to a given
site, e.g., N7, lead to a different extent of acidification, e.g., at (N1)H; the effect decreases in
the series Cu2+ > Ni2+ > Pt2+ ~ Pd2+. Moreover, the application of micro acidity constants
proves that the acidifications are reciprocal and identical. This means, Pt2+ coordinated to
(N1)–/0 sites in guanine, hypoxanthine, or adenine residues acidifies the (N7)H+ unit to the
same extent as (N7)-coordinated Pt2+ acidifies the (N1)H0/+ site. In other words, an apparently increased basicity of N7 upon Pt2+ coordination at (N1)–/0 sites disappears if the micro
acidity constants of the appropriate isocharged tautomers of the ligand are properly taken into
account. There is also evidence that proton–proton interactions are more pronounced than divalent metal ion–proton interactions, and that these in turn are possibly larger than divalent
metal ion–metal ion interactions. The indicated quantifications of the acid-base properties are
meaningful for nucleic acids including the formation of certain nucleobase tautomers in low
concentrations, which could give rise to mutations.
1. INTRODUCTION
By early 1953, “the dominant tautomeric structures that occur in the four nucleic bases of DNA” had
not yet been definitely described [1]. This deficiency caused base-pairing problems in the formulation
of the DNA double-helical structure [2]. However, since then the predominant tautomeric forms and
proton binding sites [1,3] of the common nucleobases, which also encompass uracil (being of relevance
for RNA [4]), as well as hypoxanthine, which together with the other nucleobases is an important constituent of nucleotides [5–7], have been confirmed by a variety of methods [1,4,8]. These nucleobases
are depictured in Fig. 1.
*Plenary lecture presented at the 28th International Conference on Solution Chemistry, Debrecen, Hungary, 23–28 August 2003.
Other presentations are published in this issue, pp. 1809–1919.
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H. SIGEL
Fig. 1 Predominant tautomeric forms of the six most common nucleobases [4,8].
In the upper part of Fig. 1, the three important pyrimidine nucleobases are shown; their acid-base
properties are simple, as may be exemplified with the corresponding nucleosides: In these instances, the
residues R in Fig. 1 need to be replaced by a β-D-ribofuranosyl residue to give cytidine (Cyd) and uridine (Urd), and in the case of thymine by a 2′-deoxy-β-D-ribofuranosyl residue to give thymidine
(dThd). Thus, the cytosine moiety may be protonated at N3 and the deprotonation of the (N3)H+ site in
cytidine occurs then with pK H
H(Cyd) = 4.14 ± 0.02 [9]. In contrast, uracil and thymine lose a proton from
their (N3)H site and the acidity constants for the corresponding nucleosides uridine (Urd) and thymiH = 9.18 ± 0.02 [10] and pK H
dine are pKUrd
dThd = 9.69 ± 0.03 [11,12], respectively. The error limits given
here and below refer always to three times the standard error of the mean value (3σ) or the sum of the
probable systematic errors, whichever is larger. It should also be noted that all equilibrium constants in
this article refer to 25 °C and an ionic strength, I, of 0.1 M (NaNO3) if nothing else is mentioned. To
conclude, the acid-base properties of the N3 sites of pyrimidines are well characterized and in the
physiological pH range of about 7.5 N3 of the cytidine residue is accessible for metal ion coordination
[13], whereas in the case of the uridine and thymidine residues this is only exceptionally true [10,11]
for the biologically important [14] metal ions.
For the purines shown in the lower part of Fig. 1, the situation is much more complicated. Again,
replacement, in this case of the hydrogen at N9, by a β-D-ribofuranosyl residue transfers adenine, hypoxanthine, and guanine into the nucleosides adenosine, inosine, and guanosine, respectively. However,
for example, the adenine moiety contains three basic nitrogen atoms [1,15], the most basic site, which
accepts the first proton, is N1, next, protonation occurs at N7 and then at N3 [16]. This situation is reflected in the acidity constants summarized in Fig. 2 for threefold protonated adenine (Ade) [16]; of
course, the (N9)H site may also be deprotonated with pK H
Ade = 9.8 to give the anionic adeninate [15].
Considering that, for example, in DNA N7 is exposed in the major groove and N3 in the minor
one [4,8], one would like to know the basic properties of these two nitrogen sites, yet these properties
cannot directly be measured. This is possible only for N1, since here the neutral adenine residue is protonated; the next protonation occurs at N7, but this step is of course affected by the positive charge of
the (N1)H+ site and consequently, the acidity constant pK H
H2(Ade) = –0.4 (cf. Fig. 2) does not quantify
the basicity of N7 in a neutral adenine residue. Hence, the acid-base properties of N7, and this applies
to those of N3 as well, can only be evaluated via micro acidity constants [17,18]. Such micro acidity
constants will be described below for several purine derivatives, and the effect of metal ions on deprotonation reactions of purine moieties will also be considered (Section 5).
© 2004 IUPAC, Pure and Applied Chemistry 76, 1869–1886
Intrinsic acid-base and metal ion-binding properties of purines
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Fig. 2 Acidity constants for threefold protonated adenine; for details see [16].
2. ACID-BASE PROPERTIES OF 9-METHYLADENINE AND RELATED ADENINE
DERIVATIVES
2.1 Macro acidity constants of some protonated adenines
Just like adenine itself (see Fig. 2), 9-methyladenine (9MeA) may also accept three protons. The species
H3(9MeA)3+ is protonated at N3, N7, and N1. The protons are released in this order [16,19], giving rise
to the following three equilibria where A represents 9MeA:
H3(A)3+
KH
H3(A)
2+
(A)
H2(A)2+ + H+
=
H2
[H2(A)2+][H+]/[H3(A)3+]
H(A)+ + H+
+
+
2+
KH
H2(A) = [H(A) ][H ]/[H2(A) ]
H(A)+
KH
H(A)
=
(1a)
(1b)
(2a)
(2b)
A + H+
(3a)
[A][H+]/[H(A)+]
(3b)
Deprotonation according to equilibrium 3a is expected to occur in the pH range around 4 (cf. Fig. 2),
and, indeed, this acidity constant can be measured by potentiometric pH titrations [20]. However, the
acidity constants due to equilibria 1a and 2a are accessible only by 1H NMR shift measurements or by
UV spectrophotometry [20]. The problem in these cases is that very high concentrations of a strong acid
are needed to achieve protonation of the N7 and N3 sites of an adenine derivative [16,20]. Since, for example, aqueous solutions which are 1 or 10 M in HClO4 do not have a pH of 0 or –1, respectively, but
show much higher proton activities, their pH cannot be measured with a glass electrode and a pH meter.
In those instances where the activity coefficients of perchloric acid or another strong acid differ
significantly from 1, the H+ activity may be defined and calculated according to the H0 acidity function
as originally conceived by Hammett and Deyrup [21]. We applied in our measurements [20] the listing
of Paul and Long [22], who tabulated for several strong acids in aqueous solution, including perchloric
acid, at 25 °C the H0 values in dependence on the molar concentration of the acid. It should be noted
that a 10 M HClO4 solution corresponds roughly to a H0 value of –6. In Fig. 3, a representative example of UV absorption measurements of 9MeA in the presence of varying amounts of HClO4 is shown.
The evaluation of such a series of experiments at various wave lengths is provided in Fig. 4 for the
H0/pH range of –6 to 2 [20]; the legend for Fig. 4 provides further experimental details. These measH
urements allow an estimation for K H
H3(9MeA) (eq. 1) and the determination of K H2(9MeA) (eq. 2). The
final results [20] for 9MeA regarding eqs. 1b–3b are given in entry 1 of Table 1 [23–25].
The H2(1,9DiMeA)3+ species, where 1,9DiMeA+ = 1,9-dimethyladenine, carries protons only at
the N3 and N7 sites, since N1 is methylated; consequently, in this case only equilibria 1a and 2a apply.
Similarly, in H2(1MeAdo)3+, where 1MeAdo+ = 1-methyladenosine, N3 and N7 are protonated,
whereas N1 is methylated; hence, again, equilibria 1a and 2a are the relevant deprotonation reactions.
The results [20] for these two adenine derivatives are given in entries 3 and 6 of Table 1, respectively.
© 2004 IUPAC, Pure and Applied Chemistry 76, 1869–1886
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H. SIGEL
Fig. 3 UV absorption spectra of 9-methyladenine ([9MeA] = 0.034 mM) measured in 2 cm quartz cells in aqueous
solution in dependence on the activity of H+, i.e., the H0/pH values (see also Fig. 4) were varied from H0 –5.96,
–5.07, –3.78, –2.47, –1.52, –1.08, –0.86, –0.63, –0.36, –0.10, 0.05, 0.25, 0.46 to 0.79 and pH 1.20, 1.54 to 1.88
(25 °C; I = 0.1 M, NaClO4, except in those solutions where [HClO4] > 0.1 M). Not all spectral traces of the data
points which appear in Fig. 4 are shown. Reproduced by permission of the Royal Society of Chemistry from Fig. S1
of the Supplementary Information of [20].
Fig. 4 Evaluation of the dependence of the UV absorption of 9-methyladenine ([9MeA] = 0.034 mM) at 212, 218,
220, 223, 260, and 279 nm on the activity of H+ in aqueous solution (see Fig. 3) by plotting the absorption versus
H0/pH. The evaluation of this experiment led to the weighted mean pK H
H3(9MeA) = –2.85 ± 0.36 (3σ) and to the
following individual acidity constants at the mentioned wavelengths: 212 nm, pK H
H2(9MeA) = –(0.59 ± 0.04); 218
nm, –(0.66 ± 0.03); 220 nm, –(0.67 ± 0.03); 223 nm, –(0.68 ± 0.04); 260 nm, –(0.41 ± 0.14); 279 nm, –(0.60 ±
0.19) (1σ), which gives the weighted mean pK H
H2(9MeA) = –(0.65 ± 0.09) (3σ) for this experiment. The solid curves
shown are the computer-calculated best fits at the mentioned wavelengths through the experimental data points
obtained at H0 –5.96 ([HClO4] = 10.23 M), –5.50 (9.61 M), –5.07 (9.03 M), –4.70 (8.51 M), –4.23 (7.86 M), –3.78
(7.22 M), –3.29 (6.59 M), –2.99 (6.20 M), –2.76 (5.88 M), –2.47 (5.42 M), –2.21 (4.97 M), –2.01 (4.58 M), –1.52
(3.61 M), –1.08 (2.65 M), –0.86 (2.17 M), –0.63 (1.69 M), –0.36 (1.20 M), –0.10 (0.84 M), 0.05 (0.60 M), 0.25
(0.36 M), 0.46 (0.24 M), 0.79 (0.12 M), and pH 1.20, 1.54, 1.88, 1.93, and 1.97 (from left to right) by using the
mentioned average results (25 °C; I = 0.1 M, NaNO3, except in those solutions where [HClO4] > 0.1 M).
Reproduced by permission of the Royal Society of Chemistry from Fig. 2 of [20].
© 2004 IUPAC, Pure and Applied Chemistry 76, 1869–1886
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Intrinsic acid-base and metal ion-binding properties of purines
Table 1 Negative logarithms of the acidity constants of some protonated adenine derivatives in aqueous
solution.a,b
No.
1
2
3
4
5
6
7
Adenine
derivative
Protonated
species
9-Methyladenine
N6′,N6′,N9Trimethyladenine
1,9-Dimethyladenine
7,9-Dimethyladenine
Adenosine
1-Methyladenosine
Purinek
pKa
(N3)H+
(eq. 1)
pKa
(N7)H+
(eq. 2)
pKa
(N1)H+
(eq. 3)
H3(9MeA)3+
H3(TriMeA)3+
–2.83 ± 0.30c
–2.7d
–0.64 ± 0.06c
–0.77 ± 0.13c
4.10 ± 0.01
4.18 ± 0.04
H2(1,9DiMeA)3+
H2(7,9DiMeA)3+
H3(Ado)3+
H2(1MeAdo)3+
H3(Pur)3+
–2.72 ± 0.38c
–2.63 ± 0.21c
–h
–4.02 ± 0.28c,j
<–6
–0.79 ± 0.10c
–
–1.50 ± 0.15i
–1.55 ± 0.10c
–1.7
(9.1 ± 0.1)e,f
0.50 ± 0.08g
3.61 ± 0.03
(8.69 ± 0.03)f
2.4
aThe above values are abstracted from Table 1 in [20]. The error limits, if nothing else is mentioned, are three times the
standard error of the mean value or the sum of the probable systematic errors, whichever is larger.
bSo-called practical, mixed, or Brønsted constants are listed [23]. If nothing else is mentioned, the constants were determined
by potentiometric pH titrations (25 °C; I = 0.1 M, NaNO3). In those instances where the above pKa value carries a negative
sign, I was considerably higher than 0.1 M (see the legends for Figs. 3 and 4).
cMeasured by UV spectrophotometry (Figs. 3 and 4) at 25 °C and I = 0.1 M (NaClO ) if [HClO ] < 0.1 M. The values at pH >
4
4
1 were measured with an electrode, lower ones were calculated based on the H0 scale (see text in Section 2.1).
dEstimate based on the average of entries 1, 3, and 4.
eFrom [24]; error limit estimated.
fThis value refers to the deprotonation of the (C6)NH group (eq. 4); see text and also Section 4.
2
gAverage from spectrophotometric and 1H NMR shift measurements [20].
hAdo decomposes under these conditions [16].
iAverage of the values given in [25] and [16,19]; error limit estimated.
jEstimate; see [20].
kFrom [16,19].
The acidity constants of H2(7,9DiMeA)3+, where 7,9DiMeA+ = 7,9-dimethyladenine, refer
chargewise to the deprotonation reactions 1 and 2, but it needs to be emphasized that in reaction 2 the
(N1)H+ site is deprotonated because N7 is methylated (see Fig. 6, vide infra); reaction 3 does not occur,
of course. These two acidity constants for the deprotonation of the (N3)H+ and (N1)H+ units are given
in entry 4 of Table 1. The values [20] for threefold protonated N6′,N6′,N9-trimethyladenine,
H3(TriMeA)3+ [26], which have much in common with those of H3(9MeA)3+, and for protonated
adenosine are listed in entries 2 and 5, respectively, of Table 1. Entry 7 contains the values [16,19] for
threefold protonated purine [27].
Compounds like 1,9DiMeA+ and 1MeAdo+ are known to be able to lose a proton from the
(C6)NH2 group [24,28]. The resulting zwitterionic species tautomerizes then to the uncharged imino
form [24] (see Fig. 12, vide infra in Section 4). For 1MeAdo+ the deprotonation reaction 4a holds:
1MeAdo+
(1MeAdo – H) + H+
+
+
KH
1MeAdo = [(1MeAdo – H)][H ]/[1MeAdo ]
(4a)
(4b)
The corresponding acidity constant (eq. 4b) is listed in entry 6 of Table 1 (column 6). This result will
be discussed further in Section 4.
In viewing the results of Table 1, it is interesting to see that pK H
H2(Ado) = –1.50 ± 0.15 (Table 1,
entry 5) and pK H
=
–1.55
±
0.10
(entry
6)
are
identical;
indeed,
in both instances the proton is
H(1MeAdo)
released from the (N7)H+ site in species which have overall the same charge, but in one instance the
hydrogen at (N1)H+ is replaced by a methyl group. This exchange without a significant alteration of the
acid-base properties at another nearby site is possible because of the close similarity of the electro-
© 2004 IUPAC, Pure and Applied Chemistry 76, 1869–1886
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H. SIGEL
negativity of H and CH3. This fact is also meaningful regarding the evaluation of micro acidity constants as described in Sections 2.2 and 3.
Considering the above, it is not astonishing that deprotonation of (N3)H+ in the equally charged
H3(9MeA)3+, H2(1,9DiMeA)3+, and H2(7,9DiMeA)3+ species occurs within the error limits with the
same pKa (Table 1, column 4 of entries 1, 3, and 4). Replacement of a methyl by a ribose residue at N9
leads to an acidification in all instances. This is especially evident if the pKa(N3)H and pKa(N7)H values
are compared for H2(1,9DiMeA)3+ with H2(1MeAdo)3+ (entries 3 and 6); i.e., ∆pKa(N3)H = –(4.02 ±
0.28) – (–2.72 ± 0.38) = –1.3 ± 0.5 and ∆pKa(N7)H = –(1.55 ± 0.10) – (–0.79 ± 0.10) = –0.76 ± 0.14.
The two acidifications are significant and possibly also of a similar order (note the error limits).
Substitution of a hydrogen by an amino group at C6 in purine to give adenine enhances the basicity of all N sites (cf. values in Fig. 2 with those in Table 1, entry 7), yet the additional replacement of
a hydrogen by a methyl group at C9 to give 9-methyladenine has little effect on the basicity of N1 (cf.
entry 1 in Table 1 with Fig. 2). The comparison of the acidity constants of the same two compounds for
their H3(A)3+ and H2(A)2+ species, A = Ade or 9MeA, indicates that N9 methylation reduces the basicity of N7 only slightly (if at all), but enhances that of N3 significantly [20].
2.2 Intrinsic acid-base properties of 9-methyladenine
The acidity constants (Table 1) discussed in the preceding section are macroconstants which apply to
the overall properties of a compound. Now we attempt to quantify the intrinsic acid-base properties of
a given site by applying micro acidity constants [17,18]. Figure 5 summarizes the equilibrium scheme
for H2(9MeA)2+ defining the microconstants (k) and giving their interrelation with the macro acidity
constants (K) according to the definitions provided in the lower part of Fig. 5 by following known routes
[17]. H2(9MeA)2+ may release one proton from (N7)H+ and one from (N1)H+ and therefore we rewrite
H2(9MeA)2+ as +HN7(9MeA)N1H+ or sometimes even shorter as +HN7-N1H+; since deprotonation
may occur at either site, we define the monoprotonated forms as N7-N1H+ and +HN7-N1. Figure 5
Fig. 5 Equilibrium scheme for 9-methyladenine (9MeA) (Table 1, entry 1) defining the micro acidity constants (k)
and showing their interrelation with the measured macroconstants (K) and the connection between N7-N1H+ and
+HN7-N1 and the other species. In N7-N1H+ and +HN7-N1 the proton is bound to N1 or to N7, respectively;
+HN7-N1H+ is also often written as H (9MeA)2+ or sometimes as +HN7(9MeA)N1H+. The arrows indicate the
2
direction for which the constants are defined. Once one of the micro acidity constants is known, the other
microconstants may be calculated with eqs. a–c; see also Fig. 7.
© 2004 IUPAC, Pure and Applied Chemistry 76, 1869–1886
Intrinsic acid-base and metal ion-binding properties of purines
1875
shows that there are four unknown micro acidity constants, but only three independent equations interrelating them with the macroconstants; this means, one of the microconstants needs to be obtained independently.
Figure 5 indicates that the micro acidity constants in the upper pathway are practically identical
with the measured macro acidity constants because the release of the proton from (N7)H+ is complete
before deprotonation at (N1)H+ begins. How is the situation for the lower pathway? If we recall the observations discussed in Section 2.1 that a hydrogen atom may be replaced by a methyl group without
significant alteration of the acid-base properties of nearby sites, H(7,9DiMeA)2+ with a methyl group
at N7 and a proton at N1 should mimic well the +HN7(9MeA)N1H+ species. The close similarities of
the structures of these two compounds are evident from Fig. 6. Hence, we may conclude that the
(N1)H+ site in H(7,9DiMeA)2+ represents well the corresponding site in +HN7(9MeA)N1H+, i.e.,
HN7 -N1 = pK H
pk HN7-N1
H(7,9DiMeA) = 0.50 ± 0.08. Use of this value in the lower pathway at the left in Fig. 5
H
allows us now to calculate according to the properties of a cyclic system the microconstant for the reN1
lease of the proton from (N7)H+ with N1 being unprotonated, i.e., pkN7HN 7-N1 = 2.96 ± 0.10, as summarized in Fig. 7.
Fig. 6 The above structures reveal the close similarity between H(7,9MeA)2+and H2(9MeA)2+, which allows the
HN7 -N1
2+
application of pK H
H(7,9DiMeA) = 0.50 ± 0.08 (Table 1, entry 4) for pk HN7 -N1H of H2(9MeA) in the microconstant
scheme in Fig. 5 (lower pathway at the left).
However, in developing the upper pathway in Fig. 5 correctly, one cannot simply use the macroHN7 -N1
constant pK H
H2(9MeA) = –0.64 ± 0.06 because it is relatively close to the micro constant pkHN7-N1H =
0.50 ± 0.08 (see Fig. 7); i.e., the buffer regions of the two deprotonation reactions overlap somewhat,
therefore equation (a) given in the lower part of Fig. 5 has to be used for obtaining a value for
N7 -N1H
pk HN7-N1
H/9MeA (= –0.61 ± 0.06) which appears now at the left in the upper pathway of Fig. 7. In fact,
this result allows completion of the upper cycle in Fig. 7. Hence, all micro acidity constants relevant for
the deprotonation reactions of H2(9MeA)2+ are now known and completely given in Fig. 7.
Fig. 7 Microconstant scheme for 9-methyladenine (9MeA); see Fig. 5 and its legend. Application of the value
measured for H(7,9DiMeA)2+ (see Fig. 6), pK H
H(7,9DiMeA) = 0.50 ± 0.08 (Table 1, entry 4), to the above micro
HN7 -N1 permits calculation of the other microconstants in the scheme by using eqs. a–c of
acidity constant pk HN7-N1
H
Fig. 5. The error limits of the various constants were calculated according to the error propagation after Gauss; they
correspond to three times the standard error (see also Table 1, footnote a). The microconstant scheme is redrawn
by permission of the Royal Society of Chemistry from Fig. 5 of [20].
© 2004 IUPAC, Pure and Applied Chemistry 76, 1869–1886
H. SIGEL
1876
To conclude, and this needs to be emphasized: The preceding evaluation has allowed us to quantify the acid-base properties of the adenine residue in 9MeA: (i) The intrinsic acidity constant for the
N1
H
(N1)H+ site amounts to pkN7N7-N1H = 4.07 ± 0.08; a value very close to the macroconstant pK H(9MeA) =
4.10 ± 0.01. (ii) Much more important, the micro acidity constant for the (N7)H+ site in the otherwise
N1
neutral molecule is pkN7HN 7-N1 = 2.96 ± 0.10, and it is this site which is exposed in the major groove of
DNA. (iii) Unfortunately, we are not yet in the position to provide micro acidity constants for the
(N3)H+ site in an otherwise neutral molecule, but based on indirect observations we expect the pKa
value to be on the order of about 2.
3. ACID-BASE PROPERTIES OF 9-METHYLGUANINE AND RELATED NUCLEOBASE
DERIVATIVES
In this section, we shall focus on 9-methylguanine (9MeG) as a representative of hypoxanthine- and
guanine-type compounds. 9MeG can accept protons at N3 and N7 and, in addition, it can release one
from the (N1)H site (see Fig. 1). Hence, the following three deprotonation equilibria hold, where G represents 9MeG (or another related derivative):
H2(G)2+
H(G)+ + H+
(5a)
+
+
2+
KH
H2(G) = [H(G) ][H ]/[H2(G) ]
H(G)+
G
(5b)
G + H+
(6a)
+
+
KH
H(G) = [G][H ]/[H(G) ]
(6b)
(G – H)– + H+
(7a)
H = [(G – H)–][H+]/[G]
KG
(7b)
The first proton is released from the (N3)H+ site with a very low pKa value, i.e., pK H
H2(9MeG) –1.0 (cf.
footnote c in Table 2). Reaction 6 is due to the deprotonation of the (N7)H+ site and the third proton
(eq. 7) is released from (N1)H giving rise to the anion (9MeG – H)–. Most of these macro acidity constants are accessible by potentiometric pH titrations [20]; the corresponding results for H(9MeG)+ and
some related compounds are listed in Table 2 [29–31]. These data speak for themselves and need no
further interpretation [20].
However, like in Section 2.2, one may also ask here for the intrinsic acid-base properties, e.g., of
the zwitterionic species shown in Fig. 8, which is derived from 9MeG. This type of tautomer is important when a metal ion (e.g., Pt2+) coordinates to the (N1)– site of a guanine or a hypoxanthine derivative, and then, of course, N7 may be protonated despite the metal ion at (N1)– [30,32].
We employ here the same nomenclature already used for Figs. 5 and 7 in Section 2.2. This means,
H(9MeG)+ may also be written as +HN7(9MeG)N1H and deprotonation may occur at either site giving N7-N1H or +HN7-N–. Evidently, the last mentioned tautomer is the one depicted in Fig. 8. To
measure directly the release of the proton from the (N7)H+ site of 9MeG, the (N1)– site being free, is
not possible of course, since (N1)– is much more basic than N7. However, by employing the cationic
7,9-dimethylguanine, (7,9DiMeG)+, as a mimic for H(9MeG)+, as is indicated in Fig. 9, allows us to
measure the release of the proton from the (N1)H site under conditions where N7 carries a positive
charge (Table 2, entry 2).
© 2004 IUPAC, Pure and Applied Chemistry 76, 1869–1886
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Table 2 Negative logarithms of the acidity constants of some protonated guanine and
hypoxanthine derivatives.a,b
No.
1
2
3
4
5
6
Purine
Protonated
species
pKa
(N7)H+
pKa
(N1)H0
9-Methylguaninec
7,9-Dimethylguanine
9-Methylhypoxanthine
7,9-Dimethylhypoxanthine
Inosine
7-Methylinosine
H(9MeG)+
7,9DiMeG+
H(9MeHx)+
7,9DiMeHx+
H(Ino)+
7MeIno+
3.11 ± 0.06
9.56 ± 0.02
7.22 ± 0.01
9.21 ± 0.01
6.46 ± 0.01
8.76 ± 0.03
6.20 ± 0.01
1.87 ± 0.01
1.06 ± 0.06d
a,bSee the corresponding footnotes in Table 1. The values are abstracted from Table 1 in [20] but they
are in part from earlier work [29,30].
cThe acidity constant of H (9MeG)2+ for the deprotonation of the (N3)H+ site is estimated as
2
2+
pK H
H2(9MeG) = – 1.0 ± 0.3. This constant is the average of the values given in [19] for H2(guanine) ;
+
+
since the acidity constants for the release of the protons from the (N7)H and (N1)H sites in
H(guanine)+ and H(9MeG)+ are relatively similar [30], one may assume that this is also true for the
(N3)H+ site; the given error limit is a generous estimate.
dMeasured via 1H NMR shift experiments [31].
Fig. 8 Zwitterionic form of 9MeG, i.e., (9MeG)±, sometimes written as +HN7(9MeG)N1–.
Fig. 9 The above structures emphasize the close similarity between (7,9DiMeG)+ and H(9MeG)+, which allows the
HN7 -N1
+
use of pK H
7,9DiMeG = 7.22 ± 0.01 (Table 2, entry 2) for pk HN7-N1H of H(9MeG) in the micro constant scheme in
Fig. 10 (lower pathway at the left).
Figure 10 presents the micro constant scheme for the deprotonation reactions of H(9MeG)+. It reveals that the micro acidity constants in the upper pathway are identical with the measured macro acidity constants because the release of the proton from (N7)H+ is complete before deprotonation at (N1)H
begins. Furthermore, if we use, as indicated above, the acidity constant measured for the release of the
proton from the (N1)H site in (7,9DiMeG)+ (Table 2, entry 2), then it holds for the deprotonation of
HN7 -N1 = pK H
(N1)H in +HN7(9MeG)N1H that, pk HN7
7,9DiMeG = 7.22 ± 0.01. This value given in the
-N1H
lower pathway at the left in Fig. 10 allows now to calculate according to the properties of a cyclic system the microconstant for the release of the proton from (N7)H+ with (N1)– being unprotonated, i.e.,
N1
pkN7HN 7-N1 = 5.45 ± 0.06 [20].
© 2004 IUPAC, Pure and Applied Chemistry 76, 1869–1886
H. SIGEL
1878
Fig. 10 Microconstant scheme for 9-methylguanine (9MeG) (see also Fig. 5 and its legend). Use of the value
measured for (7,9DiMeG)+ (see Fig. 9), pK H
7,9DiMeG = 7.22 ± 0.01 (Table 2, entry 2), for the above micro acidity
HN7 -N1 permits calculation of the other microconstants in the scheme (see also legend to Fig. 7). The
constant pk HN7-N1
H
above scheme is redrawn by permission of the Royal Society of Chemistry from the upper part of Fig. 4 in [20].
The corresponding micro acidity constants may also be calculated [20] for 9-methylhypoxanthine
(9MeHx) or inosine (Ino) since the corresponding 7-methyl derivatives have been studied (see Table 2).
In fact, these micro acidity constants will be employed in Section 4.
4. FORMATION DEGREES OF SOME RARE TAUTOMERS OF NUCLEOBASE RESIDUES
Micro acidity constants as described in Sections 2.2 and 3 for the (N1)H and (N7)H sites of purines are
useful and significant quantities. The values derived for H2(9MeA)2+ and H(9MeG)+ in Sections 2.2
(Fig. 7) and 3 (Fig. 10), respectively, are summarized in part in Table 3, together with the corresponding results of some other related systems. These micro acidity constants also define the intrinsic acidbase properties in aqueous solution of the corresponding individual sites in nucleic acids, at least in a
relative sense, and they can also be used to quantify in a proper way the acidifying effects of metal ions
[32] (see also Sections 5.2 and 5.3).
Table 3 Micro acidity constants for the (N7)H and (N1)H sites of several purine derivatives, together with the
ratios quantifying the formation of the connected tautomers. The measured macro acidity constants, pKa/(N7)H
and pKa/(N1)H, are given for comparison.a
No.
1
2
3
4
Purineb
pKa/N(7)H
pKa/(N1)H
N1
pkN7HN 7-N1
Ino
9MeHx
9MeG
H(9MeA)+
1.06 ± 0.06
1.87 ± 0.01
3.11 ± 0.06
–0.64 ± 0.06
8.76 ± 0.03
9.21 ± 0.01
9.56 ± 0.02
4.10 ± 0.01
3.62 ± 0.07
4.62 ± 0.02
5.45 ± 0.06
2.96 ± 0.10
N1
pkN7N7-N1H
[N7-N1H]c
[HN7-N1]
8.76 ± 0.03 138 000 ± 24 200
9.21 ± 0.01 38 900 ± 2000
9.56 ± 0.02 12 900 ± 1900
4.07 ± 0.08
12.9 ±
3.8
aFor the error limits, see footnote a of Table 1 and the legend of Fig. 7. All values apply to 25 °C and I = 0.1 M (NaNO ) with
3
the exception of the value in the third column of entry 4. The values given in columns 3 and 4 are from Tables 1 (entry 1) and 2
(entries 1, 3, 5); the micro acidity constants are taken from Table 2 in [20] (see also Figs. 7 and 10).
bAbbreviations used: Ino, inosine; 9MeHx, 9-methylhypoxanthine; 9MeG, 9-methylguanine; 9MeA, 9-methyladenine (see also
Fig. 1). The protonation degree given with the abbreviations used in column 2 corresponds to that of the tautomers which appear
in the ratio of the final column at the right.
cFor the calculation procedure, see Fig. 11.
In addition, based on such micro acidity constants (Table 3), one may calculate the ratio R* which
quantifies the formation degree of the isomeric species having the proton in one tautomer at N1 and in
the other at N7 (eq. 8):
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Intrinsic acid-base and metal ion-binding properties of purines
R* =
[N 7 - N1⋅ H] = kHN7-N1
⋅N 7-N1
N7-N1
[H ⋅ N7 - N1] kN7-N1
⋅H
1879
(8)
The procedure for such a calculation is exemplified in Fig. 11 for 9-methylhypoxanthine (9MeHx).
The results for the four systems considered here are listed in the final column of Table 3. Though
R* is somewhat sensitive to relatively small changes in the pk values involved in the calculations, it is
evident that for the guanine and hypoxanthine derivatives the neutral tautomers with the proton at N1
strongly dominate (see also Fig. 11), with formation degrees close to 100 % allowing only the formation of traces of the zwitterionic species +HN7-N1–. It is interesting to note that substitution of the
methyl group at N9 of the purine residue by a ribose moiety significantly further favors the neutral tautomer (see Table 3, entries 1 and 2). The corresponding observation has been made [20] for guanosine
(Guo) where the ratio N7(Guo)N1H/+HN7-N1– equals about 80 000 to 1, in other words, it is much
higher than the one given for 9MeG in entry 3 of Table 3 (final column). However, such rare tautomers
could well be involved in mutations of the genetic machinery since the ratio given persists in the physiological pH range (see also the terminating paragraph of this section).
For the adenine derivatives, the situation is somewhat less one-sided; here, both tautomers are
formed in appreciable amounts, e.g., monoprotonated 9-methyladenine occurs to about 93 % as the
N7-N1H+ isomer and to about 7 % in the +HN7-N1 form (Table 3, entry 4). The corresponding tautomeric distribution for adenosine [20] is 96 versus 4 %. In fact, this means that in 1 ml of a 10–3 M Ado
solution from the 6 × 1017 adenosine molecules present, about 2.4 × 1016 molecules carry the proton at
N7. However, the relatively high basicity of N7, which is reflected in the mentioned ratios, is of relevance for metal ion binding at this site [33–35], but it is not of relevance for the formation of tautomeric
species in the physiological pH range of about 7.5 since under these conditions the proton is already
lost, its pKa value being about 4 (Table 3, entry 4).
Fig. 11 Tautomeric equilibrium for 9-methylhypoxanthine between the zwitterionic, +HN7(9MeHx)N1–, and the
neutral, N7(9MeHx)N1H, forms, together with the calculation procedure for the ratio R* based on the
corresponding micro acidity constants (see also Table 3).
© 2004 IUPAC, Pure and Applied Chemistry 76, 1869–1886
H. SIGEL
1880
For adenosine, another tautomeric equilibrium, the one between an amino and an imino form, is
expected to be of more relevance in the physiological pH range since it persists under these conditions
and most likely it is also meaningful with regard to mutations occurring in nature. The tautomeric equilibrium for adenosine between its amino and imino form is displayed on the top in Fig. 12. In the context of equilibrium 4a in Section 2.2, we have already seen that 1-methyladenosine may lose a proton
from its (C6)NH2 group and that the resulting zwitterionic species stabilizes itself by tautomerizing to
the imino form as shown in the middle part of Fig. 12. By use of the corresponding acidity constants,
one may calculate [24] the ratio of the two tautomers as shown in the lower part of Fig. 12. It is evident
that the amino form of Ado strongly dominates: Among 100 000 adenosine molecules, approximately
one is present in the imino form, which is enough for initiating mutations.
Fig. 12 Tautomeric equilibrium between the amino and imino forms of adenosine. The calculation shows (for
details of the procedure, see [24]) that the amino form at the left is strongly favored. The acidity constants used in
the calculation are from Table 1.
5. EFFECT OF METAL ION COORDINATION ON THE ACID-BASE PROPERTIES OF
PURINE NUCLEOBASES
5.1 Metal ions and 9-ethylguanine
In complexation reactions of divalent and labile metal ions (M2+) with guanine derivatives in aqueous
solution, the protonation equilibria 6a and 7a of Section 3 (they are repeated below) as well as the complex-forming equilibria 9a and 10a need to be considered [30]. The species H(G)+ carries a proton at
N7, G represents the neutral guanine derivative, in this section specifically 9-ethylguanine (9EtG)
(Fig. 1), and (G – H)– the corresponding species deprotonated at N1:
H(G)+
G + H+
(6a)
+
+
KH
H(G) = [G][H ]/[H(G) ]
(6b)
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1881
(G – H)– + H+
(7a)
–
+
KH
G = [(G – H) ][H ]/[G]
(7b)
G
M2+ + G
M(G)2+
(9a)
M
KM(G)
= [M(G)2+]/([M2+][G])
M2+ + (G – H)–
(9b)
M(G – H)+
(10a)
M
+
2+
–
KM(G
– H) = [M(G – H) ]/([M ][(G – H) ])
(10b)
Of course, the complex M(G)2+ formed according to equilibrium 9a may lose a proton from its (N1)H
site to give M(G – H)+ according to equilibrium 11a:
M(G)2+
M(G – H)+ + H+
(11a)
H
KM(G)
= [M(G – H)+][H+]/([M(G)2+]
(11b)
At this point, it is important to note that equilibria 7a, 9a, 10a, and 11a are connected with each
other as is evident from the equilibrium scheme 12:
(12)
This scheme involves four equilibrium constants and because it is of a cyclic nature only three constants
are independent of each other; the size of the fourth constant is automatically determined by the other
three as follows from eqs. 13 and 14:
M
H
M
H
– pKM(G)
= log KM(G
log KM(G)
– H) – pKG
(13)
H
pKM(G)
(14)
=
pKH
G + log
M
KM(G)
M
– log KM(G
– H)
site in M(9EtG)2+
The acidification of the (N1)H
ions, may be defined by eq. 15:
complexes, as caused by the (N7)-coordinated metal
H
∆ pKa = pK H
G – pK M(G)
(15)
The corresponding results [30] for the Cu(9EtG)2+ and Ni(9EtG)2+ complexes are summarized in Table
4 (entries 2, 3).
With Pt(II), a kinetically inert metal ion, the situation is easier since it can be “fixed” at N7; it was
used in the form (Dien)Pt2+ because then only a single coordination site is left, which is tied up by N7
H
of 9EtG and consequently, pK(Dien)Pt(9EtG-N7)
(eq. 11) can directly be measured by potentiometric pH
titration [36] (Table 4, entry 4) or by 1H NMR shift experiments [32,37].
For the complexes of inosine with Pd2+ [38] and Pt2+ [39] it has been shown [30] that the acidifying properties of the two metal ions are identical within the error limits. Therefore, based on the mentioned (Table 4) plus additional results, it has been concluded [30] that the acidifying effect of (N7)-coordinated divalent metal ions on the deprotonation of (N1)H sites in guanine derivatives decreases in
the series Cu2+ > Ni2+ > Pt2+ Pd2+, the ∆pKa values being on the order of about 2.2 ± 0.3 > 1.7 ±
0.15 > 1.4 ± 0.1 1.4, respectively.
© 2004 IUPAC, Pure and Applied Chemistry 76, 1869–1886
H. SIGEL
1882
Table 4 Logarithms of the stability constants of M(G)2+ and M(G – H)+ complexes,
where G = 9-ethylguanine, as well as negative logarithms of related acidity constants as
determined by potentiometric pH titrations (aq. solution; 25 °C; I = 0.1 M, NaNO3)
together with the acidifying effect of M2+ as expressed by ∆pKa (eq. 15).a
No.
1
2
3
4
H+
or
M2+
H
pKH(G)
or
M
log KM(G)
(eqs. 6,9)
M
log KM(G
– H)
(eq. 10)
H+
Cu2+
Ni2+
Pt2+
3.27 ± 0.03
2.42 ± 0.09
1.76 ± 0.10
4.7 ± 0.4
3.48 ± 0.13
pKH
G
or
H
pKM(G)
(eqs. 7,11,14)
∆pKa
(eq. 15)
9.57 ± 0.05
7.3 ± 0.4
7.85 ± 0.17
8.17 ± 0.03
2.3 ± 0.4
1.72 ± 0.18
1.40 ± 0.06
aFor the error limits, see footnote a of Table 1 and the legend of Fig. 7. The values of entries 1–3 are
from [30]; entry 4 is from [36] and refers to the (Dien)Pt(9EtG-N7)2+ complex, where Dien =
diethylenetriamine = 3-azapentane-1,5-diamine.
5.2 The acidifying effects of metal ions at N1 or N7 are reciprocal
What is the effect of a metal ion coordinated at N7 on the (N1)H site of a guanine or hypoxanthine
residue and what is it if the same metal ion is bound at the (N1)– site on the (N7)H+ unit? These questions are intriguing and for several years the answers were confusing.
It is evident that a simple way to pursue the above problem is to employ Pt(II) because it binds in
a kinetically inert way to N1 and also to N7 of purines and consequently, the acidification on the (N1)H
and (N7)H+ sites can directly be measured [32]. We shall consider here the (Dien)Pt2+ complexes of
9-methylhypoxanthine (9MeHx) [30,32]: In (Dien)Pt(9MeHx-N7)2+ Pt(II) is coordinated to N7 of neutral 9MeHx, whereas in (Dien)Pt[+HN7(9MeHx – H)N1–]2+ Pt(II) is coordinated to the deprotonated
(N1)– site and the proton is relocated to N7 of the zwitterionic +HN7(9MeHx)N1– ligand (see also
Sections 2.2 and 3 for the nomenclature).
The acid-base properties of H(9MeHx)+ are known [20,30] (Tables 2 and 3) and those of the two
Pt2+ complexes were calculated [30] by using published 1H NMR data [40] referring to the chemical
shift in dependence on pH (pH*) in aqueous solution (D2O); the resulting values were then transformed
[30] to H2O as solvent by applying eq. 16 [41] (for details see [30]):
pKa/H2O = (pKa/D2O – 0.45)/1.015
(16)
The corresponding results are given in entries 2 and 3 of Table 5. Calculation of the acidification
according to eq. 15 for the complex with (N7)-coordinated Pt2+ leads to the result in the final column
of entry 2 and is as one would expect; i.e., Pt2+ at N7 acidifies (N1)H of 9MeHx. However, from entry
3 of Table 5, it follows that coordination of Pt2+ to the (N1)– site makes N7 more basic; therefore, the
∆pKa value carries a negative sign. In other words, replacement of H+ at (N1)– by Pt2+ makes the
(N7)H+ site less acidic compared with the situation in H(9MeHx)+; one could also say, Pt2+ is less polarizing than H+.
However, there is a caveat here which becomes evident if one compares the structures shown in
Fig. 13: The two formulas at the top refer to the comparison made between entries 1 and 2 of Table 5
giving ∆pKa = 1.54 ± 0.08, whereas the two formulas in the middle refer to the comparison between entries 1 and 3 (Table 5, column 3) giving ∆pKa = –1.15 ± 0.25. Note, the charge difference between the
two compounds seen at the top of Fig. 13 is two, whereas between the two compounds in the middle it
is only one. Hence, though the result of entry 3 in Table 5 is correct in its own right, it should not be
compared with the one given in entry 2 since the two entries do not correspond to each other.
© 2004 IUPAC, Pure and Applied Chemistry 76, 1869–1886
Intrinsic acid-base and metal ion-binding properties of purines
1883
Table 5 Acidity constant comparisons for 9-methylhypoxanthine systems with (Dien)Pt2+
coordinated either to N7 or to (N1)– (the data refer to aqueous solutions).a
No.
Acid
1b
H(9MeHx)+
2c
(Dien)Pt(9MeHx-N7)2+
3c
(Dien)Pt[+HN7(9MeHx
4d
+HN7(9MeHx)N1–
H
pK(N7)H
H
pK(N1)H
1.87 ± 0.01
9.21 ± 0.01
7.67 ± 0.08
–
H)N1–]2+
∆pKa
1.54 ± 0.08
3.02 ± 0.25
–1.15 ± 0.25
4.62 ± 0.02
1.60 ± 0.25
aFor the error limits, see footnote a of Table 1 and the legend of Fig. 7; Dien = diethylenetriamine = 3azapentane-1,5-diamine.
bI = 0.1 M, NaNO ; 25 °C. Values from [30].
3
cI 0.01–0.1 M; ambient temperature [40]. The constants were calculated [30] based on published 1H
NMR shift data [40].
dThe micro acidity constant (25°C; I = 0.1 M, NaNO ) given for the release of the proton from (N7)H+
3
under conditions where N1– is free is from [20] (see also entry 2 of Table 3, column 5). The ∆pKa value
H
values in column 3 of entries 4 and 3
given in the final column is the difference between the pK(N7)H
(see also text in Section 5.2).
In the bottom part and at the left of Fig. 13 the zwitterionic +HN7(9MeHx)N1– species is shown;
its overall charge is zero and hence, the acid-base property of this tautomer of 9MeHx needs to be compared with that of its Pt2+ complex (Dien)Pt[+HN7(9MeHx – H)N1–]2+. In other words, one has to use
in the comparison the micro acidity constant (entry 4 of Table 5, column 3) which describes the deprotonation of the (N7)H+ site for a ligand with a deprotonated (N1)– unit! This is done in entry 4 of
N1
H
Table 5, where ∆pKa is now correctly calculated as pkN7HN 7-N1 – pK Pt/complex = (4.62 ± 0.02) – (3.02 ±
Fig. 13 Structural formulas of several 9-methylhypoxanthine (9MeHx) species together with those of the
(Dien)Pt(9MeHx-N7)2+ (top; right side) and (Dien)Pt[+HN7(9MeHx – H)N1–]2+ (middle and bottom; right side)
complexes (see also text in Section 5.2).
© 2004 IUPAC, Pure and Applied Chemistry 76, 1869–1886
H. SIGEL
1884
0.25) = 1.60 ± 0.25. Now, based on the correct comparisons, the ∆pKa values of entries 2 and 4 in
Table 5 (column 5) are identical within their error limits, proving indeed that the acidifications are reciprocal as one might have expected!
To conclude, it needs to be emphasized that the above result is universal and valid not only for
hypoxanthine systems, but also for complexes involving guanine or adenine residues [32]: An acidification of a metal ion at N7 on the (N1)H0/+ site is always identical with that of a metal ion at (N1)–/0
on the (N7)H+ site!
5.3 The proton “outruns” divalent metal ions in acidifications!
We have already seen in Section 5.1 that the polarizing power and, hence, the extent of acidification differs for different metal ions, and in Section 5.2 we noted that the acidifying properties of Pt2+, if one
considers metal ion and proton binding at N1 versus N7, are reciprocal, provided the comparison is
based on the coordination of Pt2+ and H+ to the two isocharged tautomers of a given purine ligand.
However, by the use of micro acidity constants further comparisons become possible [32]. For these,
9EtG was selected here as a representative for guanines and hypoxanthines because the largest set of
data is available for this ligand.
From entry 1 in Table 6, where H+ or M2+ binding to N7 and their effect on the deprotonation of
the (N1)H site is considered, one sees again that different metal ions have a different acidifying effect
and thus, a different polarizing power (Cu2+ > Ni2+ > Pt2+). However, the largest effect is clearly
Table 6 Comparison of the effect of the proton and of several divalent metal ions on
deprotonation reactions involving 9-ethylguanine (9EtG; N7-N1H) in aq. solution.a
pKa or pka
∆pKab
+ H+
+ H+
2+NiN7-N1– + H+
2+PtN7-N1– + H+
N7-N1– + H+
7.22 ± 0.01c
7.3 ± 0.4
7.85 ± 0.17
8.35 ± 0.20d
9.57 ± 0.05
2.35 ± 0.05
2.27 ± 0.40
1.72 ± 0.18
1.22 ± 0.21
N7-N1H + H+ + H+
N7-N1–Cu2+ + H+
N7-N1–Ni2+ + H+
N7-N1–Pt2+ + H+
N7-N1– + H+
3.27 ± 0.03
(3.35 ± 0.40)
(3.90 ± 0.19)
4.42 ± 0.20f
5.62 ± 0.06c
2.35 ± 0.07
(2.27 ± 0.40)
(1.72 ± 0.20)
1.20 ± 0.21
No.
Deprotonation reaction
1a
b
c
d
e
+HN7-N1H
+HN7-N1–
2+CuN7-N1H
2+CuN7-N1–
2a
be
ce
d
e
2+NiN7-N1H
2+PtN7-N1H
N7-N1H
+HN7-N1H
+HN7-N1–Cu2+
+HN7-N1–Ni2+
+HN7-N1–Pt2+
+HN7-N1–
aFor the error limits, see footnote a of Table 1 and the legend of Fig. 7. Ni2+ or Cu2+ are coordinated
to the indicated ligand binding sites; their remaining coordination positions are occupied by H2O. In
the case of Pt2+ (entries d) the remaining three positions are occupied by Dien (= diethylenetriamine =
3-azapentane-1,5-diamine). The above data are abstracted from Table 4 in [32]. The constants refer to
25 °C and I = 0.1 M (NaNO3) [20,30,32], except for the Pt2+ systems (entries d) where I 0.01–0.1
M and ambient temperature [32,37a].
bThe difference is always calculated with the final entry e of a data set.
cMicro acidity constants taken from Fig. S9 of the Supporting Information for [20] (see also [32]).
dThe corresponding result given in entry 4 of Table 4 was determined by potentiometric pH tirations
and is certainly more exact. The above value is the result [32] of 1H NMR shift measurements [37a]
and was used here for reasons of consistency because entry 2d is from the same data set.
eThese values are given in parentheses because they have not been measured directly; they were
calculated [32] assuming “reciprocity” (see Section 5.2) by using the ∆pKa values of entry 1.
fThe calculated error limit of ±0.09 [32] was enlarged to ±0.20 because the NMR evaluation (see Fig.
S1 of the Supporting Information for [32]) is based on 11 data points, but only 4 of them are in the
critical pD range which mainly determines the pKa value.
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Intrinsic acid-base and metal ion-binding properties of purines
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achieved by the single charged proton as is also seen from entry 2 and confirmed by systems involving
9-methyladenine [32]. This illustrates the known [42] stronger polarizing power of the proton over almost any divalent metal ion; compare, e.g., the acidity of H3O+ with that of M(H2O) 2+
n ions. Overall,
these results conform to the principle [32], first formulated by Martin [43], based on results obtained
with Pd2+ complexes [38], that a proton–proton interaction is of greater magnitude than a metal
ion–proton interaction, which in turn is greater than a metal ion–metal ion interaction [36]. The generality of the last mentioned conclusion still needs to be proven.
6. CONCLUSIONS
Micro acidity constants are clearly useful data which allow to quantify the intrinsic acid-base properties of certain sites which cannot directly be measured. They also allow the calculation of the tautomeric
ratios, e.g., of monoprotonated purine-nucleobase derivatives, that is, of N7-N1H versus HN7-N1
meaning that in one isomer H+ is at the N1 site and in the other at N7 [20]. In fact, several of the nucleobase tautomers occurring in low concentration are expected to be also present in nucleic acids and
thus possibly be responsible for mutations.
Application of micro acidity constants also leads to insights into the properties of metal ion complexes [30,32]: For example, provided the intrinsic acid-base properties are considered via micro acidity
constants, it becomes evident that Pt2+ coordinated to N7 in guanine, hypoxanthine, or adenine residues
acidifies the (N1)H0/+ site to the same extent as (N1)–/0-coordinated Pt2+ acidifies the (N7)H+ unit (cf.
Section 5.2). This means that the acidification is reciprocal and the so-called increased basicity of N7
upon Pt2+ coordination to (N1)–/0 results only if comparisons are made on the basis of the macro acidity constants of the ligands (Table 5). In fact, this observation is a result of the more pronounced proton–proton interaction as compared to the divalent metal ion–proton interaction (Section 5.3) [32].
Of course, a consequence of the mentioned reciprocity is that proton binding at (N1)–/0 sites of
purines decreases metal ion binding at N7 to the same extent as proton binding at N7 decreases metal
ion binding at (N1)–/0. All these indicated results are certainly meaningful for the acid-base and metal
ion-binding properties of nucleobases in nucleic acids.
ACKNOWLEDGMENTS
Significant parts of the studies described here have been carried out within the COST D8 and D20 programmes in a most enjoyable and fruitful collaboration with Prof. Dr. Bernhard Lippert from the
University of Dortmund. His inputs and the competent technical assistance of Mrs. Rita Baumbusch and
Mrs. Astrid Sigel in the preparation of this manuscript are gratefully acknowledged. The research summarized in this article was supported by the Swiss National Science Foundation over many years. This
research is also part of the COST D20 programme and received in this context support from the Swiss
Federal Office for Education and Science.
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